Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
ATP release through pannexon channels
Gerhard Dahl
rstb.royalsocietypublishing.org
Review
Cite this article: Dahl G. 2015 ATP release
through pannexon channels. Phil. Trans. R.
Soc. B 370: 20140191.
http://dx.doi.org/10.1098/rstb.2014.0191
Accepted: 24 March 2015
One contribution of 16 to a discussion meeting
issue ‘Release of chemical transmitters from
cell bodies and dendrites of nerve cells’.
Subject Areas:
cellular biology, biophysics, neuroscience,
physiology
Keywords:
ATP, Pannexin, conductance, permeability,
potassium, allosteric
Author for correspondence:
Gerhard Dahl
e-mail: [email protected]
School of Medicine, University of Miami, 1600 NW 10th Avenue, Miami, FL 33136, USA
Extracellular adenosine triphosphate (ATP) serves as a signal for diverse
physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the
airways. ATP can be released from cells by various mechanisms. This
review focuses on channel-mediated ATP release and its main enabler,
Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel
termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has
large conductance (500 pS) and unselective permeability to molecules less
than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas
the small conformation so far has only been observed with exclusive voltage
activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux
from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both
ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the
absence of a control mechanism, this positive feedback loop would lead
to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic
receptors is provided by ATP binding (with low affinity) to the Panx1 protein
and gating the channel shut.
1. Introduction
Adenosine triphosphate (ATP) and its metabolites adenosine diphosphate
(ADP), adenosine monophosphate (AMP) and adenosine serve as transmitter
molecules in many physiological and pathological body functions [1– 3].
While the function of ATP as cellular energy store was rapidly accepted after
its discovery, the transmitter function of ATP took decades to become accepted
in the scientific community. The signalling function of ATP was first postulated
in 1929 [4] and reformulated in 1953 and 1954 [5,6]. Starting in 1970, the tenacity of Burnstock et al. [7– 9] finally led to the establishment of the field of
purinergic transmission. The field has now grown to such an extent that the
‘Purines 2014’ meeting was attended by approximately 550 researchers.
To act as a transmitter, ATP has to negotiate the plasma membrane of the
ATP releasing cell before it can act—sometimes after breakdown as ADP,
AMP or adenosine—on purinergic receptors (P1, P2X or P2Y) on a targeted
cell. Four pathways for ATP release need to be considered.
(A) Like many other transmitters, ATP can be released by exocytosis. In fact,
often ATP is co-packaged and co-released with classical transmitters, such as
acetylcholine or norepinephrine [10–13]. Further support for exocytotic/vesicular ATP release is the identification of vesicular ATP transport proteins [14,15].
Exocytosis allows for fast and targeted release of ATP onto receptors and is the
prominent if not exclusive ATP release mechanism at nerve terminals [16,17].
(B) Several observations indicate the existence of alternative ATP release
pathways. ATP is released from cells, such as erythrocytes [18], that do not contain vesicles under physiological conditions. In many cell types, there is uptake
of extracellular tracer molecules correlated with ATP release [19,20]. Several
membrane channel blockers, including carbenoxolone, can attenuate or even
completely inhibit ATP release from cells and block tracer uptake [21]. These
observations are all suggestive of a channel-mediated ATP release mechanism
allowing the passive efflux of ATP from the cell following the concentration
gradient. Actually, channel-mediated ATP release is the most ancient form [22].
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
Several membrane proteins have been presented as putative
ATP release channels, including the cystic fibrosis transmembrane regulator (CFTR) [27], the maxi anion channel [28],
connexin 43 hemichannels [29], calcium homeostasis modulator 1 (CALHM 1) [30] and pannexon channels [31]. To
serve as an ATP release channel, specific requirements have
to be fulfilled. As mundane as several of these requirements
are, they are not exhibited by some of the ATP release channel
candidates. (i) ATP must demonstrably permeate the channel.
As basic as this property gets, CFTR has been shown not to
be permeable to ATP [32]. This does not preclude a regulatory role for CFTR on a genuine ATP release channel.
However, CFTR by itself does not qualify as a permeation
pathway for ATP. (ii) The ATP-permeable channel must be
expressed in the cells from which ATP is released. Connexin
43 hemichannels have been suggested to mediate the release
of ATP in many cell types [20]. Yet, erythrocytes (and other
cells) release ATP, but do not express connexin 43 [21].
(iii) Expression of the channel must be localized at the surface
where ATP is secreted. In several polarized cells, including
airway epithelia, ATP release occurs exclusively at the
apical surface. The two connexins expressed in these cells
(Cx 30 and Cx 31), consistent with their gap junction
3. The pannexon channel
Before the discovery of pannexins (Panx1, Panx2 and Panx3),
the pharmacology of connexin hemichannels, based on a limited number of drugs, best matched ATP release in a number
of cell types. However, a series of arguments (some mentioned above) raised suspicion about connexins being
responsible for ATP release under physiological conditions.
2
Phil. Trans. R. Soc. B 370: 20140191
2. Requirements for a membrane protein
to serve as an ATP release channel
function, are exclusively expressed at the basolateral membrane and are not detectable at the apical membrane [33].
(iv) The ATP channel must be capable of being active under
physiological conditions. ATP release is involved in multiple
physiological functions that occur in a normal ionic environment and often at the resting membrane potential. Both
connexin hemichannels and CALHM 1 require removal of
extracellular calcium ions to levels that may even not occur
under pathological conditions. Furthermore, these channels
open at positive potentials, which occur in excitable cells
only for brief moments. (v) For efficient ATP release, a candidate ATP release channel should be activatable at or close to
the resting membrane potential. At this potential, an optimal
electrochemical gradient for ATP flux exists, because both
concentration gradient and electrical gradient are in the outward direction for the negatively charged ATP molecule.
(vi) The candidate ATP release channel should exhibit poor
or no charge selectivity, because dyes in the size range of
ATP of either charge can be used as a surrogate measure
for ATP release. This requirement excludes the anionselective maxi-anion channel as a ubiquitous ATP release
channel. (vii) Two arguments suggest that a candidate ATP
release channel should be activated by ATP through purinergic receptors: 1. In several tissues including astrocytes, ATP
and purinergic receptors are involved in the propagation of
calcium waves. 2. In several cells, including erythrocytes,
the phenomenon of ATP-induced ATP release is observed.
With the exception of the Panx1 channel, no experimental
evidence for an activation of a candidate ATP release channel
by ATP through purinergic receptors has been provided.
(viii) Because the activation of an ATP release channel by
ATP through purinergic receptors represents a potentially
dangerous positive feedback loop, there must be a control
mechanism preventing excessive activation of cells. In particular, a combination of the ‘death receptor’ P2X7 with an
ATP release channel would be dangerous in the absence of
a control mechanism. The simplest form of such a control
would be the inhibition of the ATP release channel by its
own permeant, ATP, involving a low affinity binding site.
Inhibition by ATP has been demonstrated for the Panx1 channel, but for no other candidate ATP release channel. (ix) The
pharmacology of the candidate ATP release channel should
match that of non-vesicular ATP release. Because of overlapping pharmacologies between the various candidate ATP
release channels, this issue has created some confusion in
the literature. However, a few drugs are of some limited
value for identification of the channel involved in ATP
release. For example, probenecid inhibits ATP release and
inhibits pannexons, but does not affect channels formed by
connexins. (x) Mutations or chemical modification of a candidate ATP release channel protein within the channel pore or
in regions controlling the channel’s patency should affect
ATP release.
rstb.royalsocietypublishing.org
(C) Because certain inhibitors of proteins mediating active
membrane transport—a prime example is the urate transport
inhibitor probenecid—were observed to interfere with ATP
release from cells, it has been suggested that transport proteins
can be involved in the release process [23]. However, that probenecid also is a strong inhibitor of the pannexin1 (Panx1)
channel [24] undermines the evidence for transport-mediated
ATP release at the plasma membrane.
(D) A fourth mechanism is only seen under pathological
conditions. Lytic release of ATP through a compromised
plasma membrane can be a consequence of direct trauma
or part of programmed cell death.
In many if not most cell types, including astrocytes, both
vesicular and channel-mediated ATP release mechanisms
coexist, complicating detailed analysis of either mechanism.
Therefore, to illuminate channel-mediated ATP release as it
occurs in astrocytes or neurons, it is imperative to elucidate
basic aspects of this process in cells where vesicular release is
absent. The mature human erythrocyte has no organelles and
no vesicles. Erythrocytes, when stimulated by a low oxygen
environment or shear stress, release ATP [18]. Thus, the erythrocyte is ideal for studying basic aspects of channel-mediated
ATP release.
The focus of this review is exclusively on channelmediated ATP release. While several membrane channel
proteins have been proposed to mediate ATP release, special
emphasis is on the pannexin1 (Panx1) channel. A broad set of
experimental evidence indicates that Panx1 mediates ATP
release under physiological and pathological conditions in
several cell types. In the plasma membrane, Panx1 appears
as a hexameric assembly of Panx1 subunits [25] to form a
transmembrane channel called pannexon [26]. Throughout
the text, this term is used to indicate the fully oligomerized
state, whereas the term Panx1 is used when reference is
made to the monomeric protein.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
The pannexon channel fulfils all criteria listed above for a
legitimate ATP release channel.
(a) Reversal potential measurements in a potassium ATP
gradient suggest high ATP permeability of pannexons in cells
expressing Panx1 exogenously [31] or endogenously [21].
Expression of Panx1 also confers stimulus-dependent ATP
release to cells [31].
(b) Cells for which channel-mediated ATP release has
been documented express Panx1, including erythrocytes,
airway epithelial cells, astrocytes, macrophages, hepatocytes,
lymphocytes, kidney tubule cells and pituitary cells. Inversely, knockdown or knockout of Panx1 expression leads to
attenuation or loss of ATP release in erythrocytes [46], astrocytes [47], airway epithelial cells [37,48], macrophages [49]
and pituitary cells [50].
(c) In polarized cells, including airway epithelial cells and
kidney tubulus cells, Panx1 co-localizes with the ATP release
site [37,38].
(d) Pannexon channels can open under physiological conditions, including regular extracellular calcium concentration
5. Release of compounds other than ATP
The pannexon’s maximal single-channel conductance is on
the order of 500 pS [31,57,58]. Flux of tracer molecules
through pannexons does not exhibit charge selectivity, as
both negatively and positively charged fluorescent dyes
applied at the extracellular surface enter cells when pannexons are open. The exact size exclusion limit for pannexons
has not been determined. However, polyethyleneglycol molecules up to 1.5 kDa [41] enter the channel to a sufficient
depth to affect channel conductance. Whether this is a
measure of the pore itself or that of an extended channel
vestibulum needs further investigation.
3
Phil. Trans. R. Soc. B 370: 20140191
4. Evidence for an ATP release function of Panx1
[34] in response to stimulation by mechanical stress, low
oxygen environment, the ligands ATP, glutamate and angiotensin II binding to their respective receptors, and increased
intracellular calcium ion concentration.
(e) Activation of pannexons by several of the aforementioned stimuli can occur at the resting membrane potential.
This property allows for efficient release of ATP downstream
of both the concentration and voltage gradients.
(f ) ATP release has been determined in many cells to correlate with the uptake of extracellular tracer molecules
(typically fluorescent dyes) that are either negatively or positively charged. The pannexon channel is permeable to both
negatively charged and positively charged dyes [21,24,51].
(g) ATP acting through either metabotropic P2Y [52] or
ionotropic P2X [53] receptors can activate pannexon channels,
enabling the phenomenon of ATP-induced ATP release and
thereby amplifying the ATP signal.
(h) The positive feedback loop provided by ATP-induced
ATP release is counteracted by a negative feedback. At high
concentrations, extracellular ATP inhibits currents through
pannexons [54]. This effect is mediated by amino acids in
both extracellular loops of Panx1 [55]. In several cell types,
including astrocytes, neurons and macrophages, Panx1 is closely associated with the purinergic receptor P2X7, and
pannexons can be opened by ATP through this association.
Several ligands to the P2X7 receptor, irrespective of whether
they act as agonist or antagonist at the receptor, inhibit
pannexon function. The affinity of the ATP binding site on
Panx1 is lower than that on the P2X7 receptor. This constellation allows for initial amplification of the ATP signal by the
positive feedback loop between pannexon and receptor.
Under physiological conditions, the amplification is limited
by the negative feedback on the pannexon. This represents
an important mechanism to preserve cell integrity, because
without it, apoptotic pathways would be activated even
with minute elevations of extracellular ATP.
(i) The pharmacology of pannexons matches well with
that of non-vesicular ATP release. Pannexons are inhibited
by a variety of compounds belonging to different chemical
groups and pharmacological classes [35]. Prime examples
are probenecid, glyburide and artemesinin. Only one compound, dipyridamole, known to affect ATP release does not
inhibit pannexons [46]. Thus, in some cells, including
mouse erythrocytes, another non-vesicular ATP release
pathway must exist.
( j) Mutations and chemical modification of Panx1 affect
ATP release, consistent with an ATP permeation pathway
provided by pannexons [56].
rstb.royalsocietypublishing.org
Because pannexins were discovered in vertebrates on the basis
of limited sequence similarity with the invertebrate innexins,
it was to be expected that the pharmacology of pannexins
would be similar to that of innexins and connexins. This
expectation led to the initial motivation to analyse the ability
of Panx1 to be an ATP release channel [31]. Indeed, most connexin and innexin gap junction inhibitors subsequently were
found to also inhibit the Panx1 channel [34,35].
Although pannexins were discovered 15 years ago as a
‘second family of gap junction proteins’ in vertebrates [36],
there still is no firm evidence for their ability to indeed
form gap junctions. Cell– cell coupling reported as a consequence of Panx1 expression could have been mediated by
connexin-based gap junction channels.
There is ample evidence to exclude a gap junction function
of pannexins, including (i) their expression in cells that do
not form gap junctions, such as erythrocytes [21]; (ii) the
expression in polarized cells only at the apical membrane
[37,38], which is not in contact with any other cell; and
(iii) the glycosylation of the pannexin proteins, which prevents
the docking of pannexons in apposed cell membranes [25,39].
Unquestionably, Panx1 forms a patent non-junctional
membrane channel that allows the exchange of molecules
smaller than 1.5 kDa between intra- and extracellular space
[21,31,40,41]. (Of the three pannexins, only the properties of
the Panx1 channel are sufficiently characterized for inclusion
in this discussion.) Despite the lack of sequence homology
to connexins and the very limited homology to innexins,
pannexins fold in a similar way: both amino- and carboxytermini are located intracellularly, four segments traverse
the plasma membrane and two loops face the extracellular
space [21]. Six Panx1 subunits oligomerize [25] to form a pannexon channel in the non-junctional plasma membrane [26].
The channel properties are unusual, and, as shown below,
vary with the mode of stimulation.
Innexins are bifunctional, as they can form gap junction
channels as well as non-junctional membrane channels, innexons. The properties of innexons as far as tested are identical to
pannexon properties [42–45].
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
6. Expression and function of pannexons
in the central nervous system
7. Activation of pannexons in physiological
and pathological conditions
An experimentally convenient way to activate pannexons is
by depolarizing voltage steps applied to cells. However, the
voltages required for channel activation are in the positive
range and thus do not occur physiologically except in excitatory cells for a brief period during the peak of an action
potential. Nevertheless, a number of physiological stimuli
for pannexons have been identified that open the channel at
the resting membrane potential.
4
Phil. Trans. R. Soc. B 370: 20140191
At present, it is uncertain whether the release of ATP and
similar-sized molecules, such as glutamate and other transmitters, is the exclusive function of pannexons. Nevertheless, there
is accumulating evidence that pannexons are involved in multiple functions of the central nervous system (CNS). This is
indicated by the apparent involvement of pannexons in pathological states such as epilepsy [60], migraine [61], hypoxic
depolarization [57], Crohn’s disease with its neuronal loss in
the enteric nervous system [62] and neuroinflammation [63].
Panx1 is widely expressed in the body, including the brain.
Panx2, which is essentially uncharacterized in terms of channel
properties and functional roles [64], is almost exclusively
expressed in the CNS. Expression of Panx1 includes astrocytes
and neurons [65]. In neurons, it appears that Panx1 may exist in
different conformations or associations with other proteins.
Immunohistochemical analysis of Panx1 expression has
shown that different validated anti-Panx1 antibodies stain
different parts of neurons [66]. The most likely interpretation
of this phenomenon is epitope masking or unmasking by
either differential protein folding, differential post-translational
modification or differential interaction with other proteins in
cell bodies and dendrites.
Several intimate interactions between Panx1 and other proteins in astrocytes and neurons have been identified. A direct
physical interaction is indicated by co-immunoprecipitation
for Panx1 and the P2X7 receptor [63] or the potassium channel
subunit Kvb3 [67]. Furthermore, Panx1 can be activated by glutamate binding to NMDA receptors, by noradrenaline through
alpha adrenergic receptors, or by angiotensin II binding to AT1
receptors [68–70]. The interaction of Panx1 with Kvb3 is intriguing, as the pharmacology of the pannexon is changed by
this interaction. The inhibitory effect of carbenoxolone, probenecid and reducing agents was attenuated in co-expressing
cells when compared with the effect in cells expressing Panx1
alone. In the CNS, Panx1 and Kvb3 co-localize in pyramidal
neurons in the hippocampus and in Purkinje cells in the cerebellum [67]. The specific functional role of this co-expression
is not known.
(a) Pannexons are mechanosensitive and can be opened at
250 mV in excised membrane patches by application of negative pressure applied to the patch pipette [31]. This channel
property could be the basis for the observed swelling-induced
ATP release from erythrocytes [21] and airway epithelial cells in
response to hypotonic stress [37,48]. Activation of pannexons in
response to cell swelling has also been observed in other cell
types, including lens [71], neurons [72], astrocytes [73],
bovine ciliary epithelial cells [74] and fibrosarcoma cells [75].
(b) Low oxygen elicits pannexon-mediated ATP release
from erythrocytes [21,76] and neurons [57]. In neurons, low
oxygen could be sensed via the orthodox mitochondrial pathway, which in turn activates pannexons. In the organelle-free
erythrocyte without mitochondria, a more direct activation of
pannexons must exist. Because the activity of the potassium
channel slo1 BK can be modulated by haem [77], a similar
mechanism may control pannexon activity in erythrocytes
in an (oxygen)-dependent way.
(c) Increase in cytoplasmic calcium ion concentration
([Ca2þ]i) has been shown to activate pannexons in excised
inside–out membrane patches [52]. Consistent with this activation mechanism, pannexons are activated by ligands
binding to metabotropic receptors signalling through IP3
and [Ca2þ]i. Receptor-mediated pannexon activation has
been shown for P2Y receptors [52,78] and angiotensin II
receptors [70]. Buffering intracellular Ca2þ with 1,2-bis(oaminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA)
prevents activation of pannexons by angiotensin II and ATP
through their respective receptors [70,78]. This observation
lends further support to the notion that increases in [Ca2þ]i
can activate pannexons. Because each point has its counterpoint, it should be noted that induction of pannexon
currents by increased [Ca2þ]i can also be missed [79].
(d) Pannexons can be opened by ATP binding to purinergic
receptors. In addition to the aforementioned activation through
the metabotropic P2Y receptors, binding of ATP to ionotropic
P2X7 receptors also leads to pannexon activity [51,53]. It
appears that some other P2X receptors have the same capability [80]. While it is conceivable that the Ca2þ influx
through a P2X receptor can trigger pannexon activation, this
process is not needed, because removal of extracellular calcium
does not attenuate the ATP-induced pannexon current [53].
Thus, other signalling events must be involved. Because
Panx1 can be immunoprecipitated with P2X protein by
antibodies directed to either Panx1 or P2X [63,80,81], an activation of pannexons by direct protein–protein interaction is a
distinct possibility. In addition (or instead), another signalling
pathway appears to be involved the activation mechanism.
Pannexon currents in cells co-expressing Panx1 and P2X7R
are attenuated by inhibition of Src tyrosine kinases by a TATP2X7 peptide representing the death signal domain in the
COOH terminus of P2X7R, by the P2X7R blocker KN-62 or
by the Src tyrosine inhibitor PP2 [82].
While the identification of the molecular components
involved in ATP-induced ATP release is recent, the phenomenon as such has long been known [83– 85]. Considering that
ATP at high concentrations in the extracellular medium leads
to cell death, this positive feedback loop for ATP release is
potentially dangerous. Under physiological conditions, the
negative feedback in form of pannexon inhibition by extracellular ATP counteracts the ATP-induced ATP release and
thereby limits overstimulation of apoptotic/pyroptotic
pathways [86].
rstb.royalsocietypublishing.org
Nevertheless, based on the large pore size and the apparent
lack of selectivity, it can be expected that molecules in the size
range of ATP will pass through pannexons without hindrance.
The signalling molecules uridine 50 -triphosphate (UTP) and
glutamate fall into this category. Furthermore, it appears that
epoxyeicosatrienoic acids permeate pannexons [59].
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
8. Two channel conformations of Panx1
A preponderance of evidence designates pannexons as the
main conduit for efflux of ATP from many cell types
(reviewed in references [26,86,101]). Nonetheless, recent independent studies have shown the pannexon to be a highly
selective chloride channel with no ATP permeability
[102,103]. Furthermore, in these studies, the pannexon exhibited a unitary conductance of 74 [103] or 68 pS [102] rather
than exhibiting the complex gating behaviour and maximal
conductance of 500 pS reported earlier [21,31,52,57].
Chloride selectivity of pannexons in the Ma et al. [102] and
Romanov et al. [103] studies is indicated by two basic observations. Replacement of extracellular Naþ by NMDGþ had no
effect on pannexon currents. Replacement of extracellular Cl2
by gluconate strongly attenuated the currents and replacement
by glutamate and aspartate abolished pannexon currents
altogether. Furthermore, no ATP release from cells overexpressing Panx1 was observed despite robust pannexon currents.
Several explanations could account for the discrepancies
in the reported channel conductances and permeabilities of
pannexons. For example, different cell types with differing
5
Phil. Trans. R. Soc. B 370: 20140191
gradients and loss of energy necessary for cell survival.
Indeed, excessive or irreversible activation of pannexons
leads to cell death [53,63,94,95]. The self-inhibition of pannexons by ATP is a component of fine tuning of a potentially
dangerous channel by preventing prolonged opening.
(n) Cleavage of Panx1 by caspase 3 at its cognizant site in the
carboxyterminal sequence of the protein irreversibly activates
pannexons, invariably killing the cells [95]. This type of activation occurs in only a select group of cells and only after
these cells are committed to apoptotic cell death. The expected
ensuing ATP release mobilizes macrophages to the site of cell
death. ATP in this setting has been termed the ‘find me
signal’. A similar phenomenon also occurs in brain lesions,
where ATP initiates the response of microglia to an injury [96].
In a leech model for nerve injury, which is amenable to a rigorous analysis of molecular events, the signalling responsible for
migration of microglia to a lesion is complex. Directional movement and accumulation of microglia involve at least three
signals: the ATP released at the lesion site is the ‘go’ signal,
NO is the ‘where’ signal [97] and ArA is a ‘stop’ signal [44].
Caspase cleavage of Panx1, however, is not an obligatory
event in the anoxic depolarization that precedes death of
neurons [87]. Western blots of hippocampal slice preparations
show only the uncleaved form of Panx1 under conditions that
open pannexons. In addition, pharmacology shows that
anoxia-induced pannexon activation is caspase-independent.
Published data do not show whether the caspase 3 cleaved
pannexon is permeable to ATP. Single-channel recordings of
pannexons under conditions where the caspase-cleaved form
should prevail show a unitary conductance of 75 pS [98] or
even as low as 20 pS [99]. As shown below, the low conductance pannexon is not permeable to ATP. Either the patches
with the low conductance pannexon did not happen to contain the fully activated, ATP-permeable configuration or the
caspase-cleaved Panx1 does not form pannexons with ATP
permeability. The latter interpretation is also suggested by a
lack of reversal potential shift associated with caspase 3 cleavage of Panx1 [95,99,100]. In contrast, the Kþ-stimulated
pannexon is ATP permeable [31,58] and exhibits a large
shift in the reversal potential [47].
rstb.royalsocietypublishing.org
(e) Glutamate binding to NMDA receptors activates pannexons [68]. This process is thought to be responsible for
anoxic depolarization of pyramidal neurons, as occurring in
stroke. Intriguingly, the signalling between receptor and pannexon is similar to that described for the P2X7/pannexon
interaction. While Ca2þ influx through the NMDA receptors
in principle could be sufficient for the activation process,
experimental evidence here also points to an important role
of Src kinases in the activation of pannexons [87].
(f ) Pannexons can be activated by agonists of the alpha
1D adrenergic receptor [69]. This amplifies the adrenergic
signalling cascade enhancing the vasoconstrictive effect of
adrenergic agonists in resistance vessels. The mechanism
of activation of pannexons through alpha adrenergic receptors is not yet known. Because alpha adrenergic receptors
typically signal through phosphatidylinositol– IP3–Ca2þ,
pannexons might be directly stimulated by increased
[Ca2þ]i, although other possibilities remain [88].
(g) Thrombin binding to its receptor increases [Ca2þ]i and
stimulates release of ATP [89]. Pre-loading the cells with
BAPTA attenuates this, as if the increase in [Ca2þ]i were
directly responsible for pannexon opening and ATP release.
While pharmacological evidence indicates that the RhoA/
ROCK pathway is also activated by the thrombin receptor
activation [48], it is not known if this pathway is obligatory.
(h) Bradykinin-induced increase in [Ca2þ]i involves pannexon activity and P2Y receptor activation, as indicated by
pharmacological evidence. Whether the initial increase in
[Ca2þ]i is the primary stimulus for pannexons, which then
activate P2Y receptors to further amplify the signal, is not
clear [90].
(i) Histamine-induced ATP release seems to follow the
same pattern as described for bradykinin [91].
( j) Angiotensin II-induced ATP release is inhibited by
intracellular calcium chelation with BAPTA and thus pannexon activation could be a direct consequence of the rise
in intracellular [Ca2þ] [70].
(k) Thromboxane receptor-mediated activation of pannexons, in contrast, involves the cAMP ! protein kinase A
pathway [92].
(l) Regulation of catecholamine release from adrenal chromaffin cells apparently involves pannexon activity and ATP
release, which in turn amplifies the calcium signalling triggered by nicotinic receptors [93].
(m) Increases in extracellular Kþ concentration ([Kþ]o) stimulates pannexon-mediated ATP release [63]. The dose–response
curve shows a steady increase in ATP release from 10 mM
[Kþ]o to at least 150 mM [Kþ]o [47,58,63]. Several independent
observations indicate that [Kþ]o acts on pannexons. Xenopus
oocytes expressing Panx1 de novo acquire Kþ-stimulated ATP
release. KO/knockdown of Panx1 abolishes Kþ-induced
ATP release in astrocytes. The Kþ-induced pannexon activation
is not an effect of depolarization as judged by the voltage dependence of pannexons. Indeed, [Kþ]o activates pannexons directly
at any membrane potential in the range 2100 to þ100 mV under
voltage clamp conditions (see below).
Activation of pannexons by all the aforementioned stimuli
is rapidly reversible. For many physiological functions involving pannexons, such as platelet activation, oxygen supply,
hearing, airway cilia beat regulation, quick reversibility is indispensable. Considering pannexons’ large conductance and the
permeability to all molecules up to 1.5 kDa, prolonged activation of pannexons would lead to the rundown of ionic
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
(a)
Ofull
6
rstb.royalsocietypublishing.org
C
10 pA
(b)
5s
O2
O1
C
Figure 1. Single-channel currents through Panx1 channels with Kþ or Naþ
in the extracellular solution. (a) A pannexon in an inside –out membrane
patch exposed to high extracellular [Kþ] and clamped at – 100 mV exhibited
a maximal conductance of approximately 500 pS. The fully open and fully
closed states are indicated by red lines; the dashed lines indicate levels of
three major subconductances. Both the bath and pipette solutions contained
140 mM KGlu, 10 mM KCl and 5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.5). (b) An outside – out patch exposed to
a low extracellular [Kþ] (Ringer solution) and clamped at þ50 mV containing two channels that opened to the current levels O1 and O2. Both the
pipette and bath solutions contained 140 mM NaCl, 10 mM KCl and 5 mM
TES ( pH 7.5). As shown previously, the conversion between small and
large channel conductance can be observed in the same channel in an
outside – out patch by switching between normal Ringer and high Kþ in
the extracellular solutions [58]. (Online version in colour.)
during repolarization when some of the channels are still
open and both concentration and voltage gradients are
directed outwards [104]. For the same reasons, one cannot
expect a robust influx of positively charged YoPro (4-[(3methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3-(trimethylammonio)propyl]quinolinium diiodide) against the strong
electrical gradient of positive voltages required to open
pannexons by voltage alone [105].
That different stimuli trigger two conformations of pannexons is also indicated by differential thiol reagent accessibility.
Panx1 has a carboxyterminal cysteine, which can be reacted
with thiol reagents in the voltage-activated state of the channel
[56]. In contrast, pannexons opened with extracellular Kþ
cannot be modified by thiol reagents [58]. These observations
suggest that the inner (cytoplasmic) aspect of the pannexon
assumes drastically different conformations with different
stimuli. Cysteines replacing autochthonous amino acids in
the outer (extracellular) portion of pannexon channel permeation pathway can be reacted with thiol reagents with
either stimulation modality. Thus, the pore structure at the
extracellular side of the channel is similar or identical with
different stimuli. Figure 3 illustrates schematically the two
conductance/permeability states of pannexons.
9. Allosteric effect of Kþ on Panx1
Several lines of evidence indicate that the opening of pannexons by extracellular Kþ is due to a direct allosteric effect.
Under voltage clamp conditions, Kþ activates pannexons
Phil. Trans. R. Soc. B 370: 20140191
modulators of pannexons could be responsible for the differing channel properties [101,103]. Alternatively, Panx1 may
serve as a regulator of the actual ATP release conduit.
Scrutiny of the experimental conditions used in the various studies, however, reveals marked discrepancies.
Notably, the low conductance, ATP-impermeable pannexons
were observed with the channels activated exclusively by voltage. In contrast, the high conductance, ATP-permeable
pannexons were observed with different physiological/
pathological channel stimuli. A systematic analysis of this
phenomenon revealed that, depending on the stimulus
modality, pannexons assume different conformations with
different conductances and permeabilities [58].
The exclusively voltage-gated pannexon is not permeable
to ATP. Measuring ATP release from voltage-clamped oocytes
with a luciferase assay revealed that no pannexon-mediated
release occurred despite robust voltage-induced pannexon
currents [58]. Increasing [Kþ]o under otherwise identical conditions resulted in an ATP release of the same magnitude
as observed in Kþ-stimulated unclamped oocytes. The Kþstimulated ATP release was independent of the membrane
holding potential in the range from 260 to þ40 mV.
The two permeability states of pannexons correlate with the
two conductance states of the channel. The previously published differences in conductance states were found in
different cell types, opening the possibility for cell-type-specific
properties of pannexons. However, the two conductance states
can be seen in the same cell and even in the same channel contained in an excised outside–out membrane patch [58].
A simple change of the extracellular ion composition induces
the change in conductance. With Naþ in the extracellular solution, the channel opens only at positive potentials as also
seen for whole cell currents. The unitary conductance under
this condition is about 50 pS and thus in the same range as
reported for other cell types under comparable experimental
conditions [102,103]. Replacing extracellular Naþ with Kþ
opens pannexons in the voltage range from 2100 to
þ100 mV, also consistent with macroscopic currents. In high
[Kþ]o, the pannexon currents are complex, indicating the existence of several subconductance states and a maximal
conductance of ca 500 pS (figure 1). The large pannexon conductance and/or ATP permeability have been observed in
several cell types with different physiological or pathological
stimuli. This includes Kþ-activated pannexons in oocytes
expressing Panx1, erythrocytes and astrocytes, [31], lowoxygen-stimulated neurons or erythrocytes [21,57], mechanically stressed pannexons in oocytes or erythrocytes [21,31],
intracellular calcium-induced pannexon activity in oocytes
[52], ligand–receptor activation mechanisms of pannexons
listed above, some possibly by elevated [Ca2þ]i or other signalling chains. Thus, Kþ-activation of pannexons mimics that by
physiological activators of the channel. Kþ can therefore be
used as a surrogate for the physiological activators. For experiments, Kþ-stimulation is considerably more convenient than
some of the physiological stimuli (figure 2).
In retrospect, it is clear that the search for ATP permeability in the exclusively voltage-gated pannexon would
be futile. An unfavourable voltage gradient at high positive
membrane potentials limits or even prevents ATP efflux
from cells following the concentration gradient. For example,
no ATP efflux through connexin hemichannels, which open
only at extreme positive voltages, is measurable at þ80 mV,
but ATP release occurs briefly during the tail currents
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
7
Cl
–
+
50 pS
500 pS
physiological stimulation of Panx1 channels
1.
2.
3.
4.
5.
6.
7.
8.
exclusive activation by voltage
mechanical stress
low oxygen
intracellular [Ca2+]
ATP through purinergic receptors (P2Y and P2X)
glutamate through NMDA receptors
noradrenaline through alpha-adrenergic receptors
angiotensin 2 through AT1 receptors
activation of thromboxane receptors
experimentally and under pathological conditions
9. extracellular K+
?
cleavage of COOH-terminus by caspase 3
?
Figure 2. Permeability and conductances of pannexons vary with the activation mode. The pannexon has high conductance and is permeable to ATP, when activated by various physiological stimuli or by elevated extracellular [Kþ]. Activation of pannexons only by stepping the membrane potential to positive values results
in a small channel conductance and selective permeability to Cl2. It is unclear which channel figuration is assumed by the caspase 3 cleaved pannexon. No direct
measurements of ATP permeability of the caspase-cleaved pannexon are available, and the single-channel conductance has been reported to be 20 or 75 pS. (Online
version in colour.)
over a wide voltage range (2100 to þ100 mV). Various ion substitutions do not interfere with the Kþ effect. Kþ stimulates
pannexons in diverse cell types, including oocytes, erythrocytes and astrocytes, making it unlikely that another protein
is required. The Kþ-mediated activation is observed in excised
membrane patches containing a pannexon. The most compelling evidence for a direct action of Kþ on Panx1 comes from
electron microscopic (EM) studies. With a soluble factor (Kþ),
it is possible to activate pannexons assembled from purified
Panx1 protein in vitro and visualize the effects with negative
stain EM techniques. The Sosinsky laboratory showed that
Kþ changes the diameters of the extra- and intracellular
vestibula of pannexons by 30% and 300%, respectively [58].
10. ATP and Kþ entangle on pannexons
The initial determination of ATP permeability of pannexons
has been performed on excised membrane patches exposed
to a 10 : 1 gradient of potassium ATP. ATP permeability in
this experiment was indicated by the reversal potential being
ca 20 mV shifted from the potassium equilibrium potential.
Considering the inhibitory effect of ATP on pannexons and
the use of high concentrations of ATP in the extracellular
aspect of the membrane patch, this experiment should not
have worked. A reassessment of the effect of ATP on
pannexons in different ionic conditions, however, revealed
that the inhibitory effect of ATP on pannexons is attenuated
in a dose-dependent way by increased extracellular Kþ.
The interplay of Kþ and ATP can be observed directly in
Panx1-expressing oocytes [94]. Membrane currents induced
by depolarization are inhibited by ATP and more effectively
by BzATP (benzoyladenosinetriphosphate). Increasing extracellular [Kþ] attenuates or abolishes the inhibitory effect of
ATP and BzATP depending on the [Kþ] and [BzATP] used,
as if the two compounds compete at overlapping binding
sites. Consistent with this, Kþ also attenuates the inhibitory
effect on pannexons of BB FCF (brilliant blue for colouring
food) and Fast Green, which have an overlapping binding
site on Panx1 with that of ATP [106].
11. Pannexons can mediate cell death
The entanglement between ATP and Kþ on pannexons can
also be observed in astrocytes and in brain slices as an
approximation of in vivo conditions [94]. Astrocytes exposed
to high [Kþ]o (50 mM) release lactate dehydrogenase (LDH),
indicating cell death. LDH release is abrogated by either
blocking pannexon activity pharmacologically or by Panx1
knockout. Knockout of the P2X7 receptor does not affect
Kþ-induced LDH release. At a moderate [Kþ]o (25 mM),
Phil. Trans. R. Soc. B 370: 20140191
+
–
rstb.royalsocietypublishing.org
ATP
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
closed channel
K+-activated channel
500 pS
ATP
–
+
K+
K+
ATP
+
–
ATP
Figure 3. Cartoon of pannexon activation by different stimuli. In the unstimulated pannexon (centre), the channel pore is occluded and the terminal cysteine is not
accessible to thiol reagents. Activation by stepping the membrane potential to positive values renders the terminal cysteine reactive to extracellularly applied thiol
reagents. The single-channel conductance under this condition is circa 50 pS (left). Activation of pannexons by elevated extracellular [Kþ] at any membrane potential yields a channel with high conductance (500 pS; right). In this state, the terminal cysteine (yellow dot) is not sensitive to thiol reagents, while an engineered
cysteine at the extracellular vestibulum is. In high extracellular [Kþ], the pannexon is permeable to ATP and likely has the same configuration as pannexons activated
by various physiological stimuli. (Online version in colour.)
low BzATP (a potent ATP analogue for pannexon inhibition) enhances (through P2X7) and high BzATP attenuates
LDH release from astrocytes, consistent with the Kþ/ATP
interaction at the pannexon observed in oocytes.
Similarly, activation of the ‘executioner’ caspase 3 by Kþ
is inhibited at low [Kþ]o, but not at high [Kþ]o, by BzATP.
Caspase 3 activation is absent in Panx1 KO cells but is unaltered in P2X7 KO cells. This shows that Panx1 is upstream of
caspase 3 in the apoptotic signalling chain. Thus, Panx1 is
both an activator and a downstream substrate of caspase 3,
because as pointed out above, cleavage of Panx1 by caspase
3 can activate pannexons.
Yet another indicator of apoptotic cell death, phosphatidylserine (PS) exposure on the external leaflet of the plasma
membrane is stimulated by Kþ in a Panx1-dependent way.
Annexin binding as a measure of PS exposure is stimulated
by Kþ in wt astrocytes, but not in Panx1 KO astrocytes. With
low Kþ stimulation, BzATP attenuates annexin binding, as predicted by the interplay between Kþ and ATP on pannexons
observed in oocytes.
Additionally, in situ blockade of pannexons by ATP as
determined by dye uptake in brain slices is present in wt mice
and P2X7 KO mice, but not in Panx1 KO mice. In the absence
of the oocyte data, one would be hard pressed to interpret the
data obtained in cultured astrocytes and brain slices.
The interplay between ATP and Kþ at pannexons has profound consequences in pathological conditions where
damaged cells spill a series of pannexon activators onto adjacent healthy neighbouring cells [86,94]. Among these
activators is Kþ, which is a direct pannexon stimulant and
also abolishes the negative feedback control on pannexonmediated ATP release. In this constellation, the unopposed
positive feedback for ATP release will result in overstimulation of P2X purinergic receptors and, owing to their linkage
to the inflammasome, cause apoptotic/pyroptotic death of
cells not damaged by the original insult. This phenomenon
has long been known and is termed secondary cell death.
Typically, secondary cell death makes the lesion volume several fold larger than the primary lesion. Because this process
extends over several hours, there is a therapeutic window to
limit secondary cell death and Panx1 is a logical target for
such intervention.
12. What does it all mean?
Since the discovery of the pannexins in 2000 [36], 460 papers (as
of December 2014) have been dedicated to this small family of
‘gap junction’ proteins comprising three members. Rather than
forming the cell-to-cell channels of authentic gap junction proteins that the innexins and connexins do, pannexins apparently
form exclusively non-junctional membrane channels. These
pannexons allow the exchange of small molecules between
the intra- and extracellular spaces. While the repertoire of
potential permeants is large, only the permeation of ATP
through pannexons has been studied in detail.
In several physiological settings, ATP release through
pannexons is the primary event, i.e. a stimulus activates pannexons. Direct pannexon-mediated ATP release is found for
example in erythrocytes exposed to low oxygen or mechanical stress, airway epithelial cells stimulated mechanically
and also tubular cells in the kidney.
In several activation schemes of pannexons, the ensuing
ATP release is a secondary event with exclusive amplification
function for the signalling cascade initiated by the initial
receptor ligand. This applies to all presently known ligandmediated pannexon activations. In the case of ATP, it is a
self-amplification of purinergic signalling. For the other
ligands, pannexon-mediated ATP release amplifies other signalling cascades, as shown for alpha-adrenergic, histamine,
thrombin and angiotensin II signalling.
It is possible that pannexons exert other functions unrelated to the release of ATP, glutamate and other compounds
Phil. Trans. R. Soc. B 370: 20140191
ATP
ATP
8
rstb.royalsocietypublishing.org
voltage-activated channel
50 pS
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
expression of Panx3 is restricted to skin and bone [110], excluding
a general function comparable to that of Panx1. Thus, the challenge to identify physiological functions for Panx2 and Panx3
remains and some inspired approaches will be required.
Acknowledgements. The critical reading of the manuscript by Dr Kenneth
Muller is very much appreciated.
Funding statement. The author thanks the Craig Nielsen Foundation for
support. All applications related to the work by the author described
here were triaged by NIH study sections.
Competing interests. The author has no competing interests.
1.
Burnstock G. 1990 Overview. Purinergic mechanisms.
Ann. N.Y. Acad. Sci. 603, 1–17; discussion 8. (doi:10.
1111/j.1749-6632.1990.tb37657.x)
2. Burnstock G. 1995 Current state of purinoceptor
research. Pharm. Acta Helv. 69, 231 –242. (doi:10.
1016/0031-6865(94)00043-U)
3. Burnstock G. 2008 Purinergic signalling and
disorders of the central nervous system. Nat. Rev.
Drug Discov. 7, 575–590. (doi:10.1038/nrd2605)
4. Drury AN, Szent-Gyorgyi A. 1929 The physiological
activity of adenine compounds with especial
reference to their action upon the mammalian
heart. J. Physiol. 68, 213–237. (doi:10.1113/
jphysiol.1929.sp002608)
5. Holton FA, Holton P. 1953 The possibility that ATP is
a transmitter at sensory nerve endings. J. Physiol.
119, 50P –51P.
6. Holton FA, Holton P. 1954 The capillary dilator
substances in dry powders of spinal roots; a
possible role of adenosine triphosphate in chemical
transmission from nerve endings. J. Physiol. 126,
124–140. (doi:10.1113/jphysiol.1954.sp005198)
7. Burnstock G, Campbell G, Satchell D, Smythe A.
1970 Evidence that adenosine triphosphate or a
related nucleotide is the transmitter substance
released by non-adrenergic inhibitory nerves in the
gut. Br. J. Pharmacol. 40, 668 –688. (doi:10.1111/j.
1476-5381.1970.tb10646.x)
8. Burnstock G. 1971 Neural nomenclature. Nature
229, 282–283. (doi:10.1038/229282d0)
9. Su C, Bevan JA, Burnstock G. 1971 [3H]adenosine
triphosphate: release during stimulation of enteric
nerves. Science 173, 336 –338. (doi:10.1126/
science.173.3994.336)
10. MacKenzie I, Burnstock G, Dolly JO. 1982 The effects
of purified botulinum neurotoxin type A on
cholinergic, adrenergic and non-adrenergic,
atropine-resistant autonomic neuromuscular
transmission. Neuroscience 7, 997 –1006. (doi:10.
1016/0306-4522(82)90056-2)
11. Wagner JA, Carlson SS, Kelly RB. 1978 Chemical and
physical characterization of cholinergic synaptic
vesicles. Biochemistry 17, 1199– 1206. (doi:10.
1021/bi00600a010)
12. Silinsky EM. 1975 On the association between
transmitter secretion and the release of adenine
nucleotides from mammalian motor nerve
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
terminals. J. Physiol. 247, 145–162. (doi:10.1113/
jphysiol.1975.sp010925)
Unsworth CD, Johnson RG. 1990 Acetylcholine and
ATP are coreleased from the electromotor nerve
terminals of Narcine brasiliensis by an exocytotic
mechanism. Proc. Natl Acad. Sci. USA 87, 553– 557.
(doi:10.1073/pnas.87.2.553)
Sawada K, Echigo N, Juge N, Miyaji T, Otsuka M,
Omote H, Yamamoto A, Moriyama Y. 2008
Identification of a vesicular nucleotide transporter.
Proc. Natl Acad. Sci. USA 105, 5683 –5686. (doi:10.
1073/pnas.0800141105)
Larsson M, Sawada K, Morland C, Hiasa M,
Ormel L, Moriyama Y, Gundersen V. 2011
Functional and anatomical identification of a
vesicular transporter mediating neuronal ATP
release. Cereb. Cortex 22, 1203– 1214. (doi:10.1093/
cercor/bhr203)
Sperlágh B, Vizi SE. 1996 Neuronal synthesis,
storage and release of ATP. The Neurosciences 8,
175 –186. (doi:10.1006/smns.1996.0023)
Edwards FA, Gibb AJ. 1993 ATP: a fast
neurotransmitter. FEBS Lett. 325, 86 –89. (doi:10.
1016/0014-5793(93)81419-Z)
Bergfeld GR, Forrester T. 1992 Release of ATP from
human erythrocytes in response to a brief period
of hypoxia and hypercapnia. Cardiovasc. Res. 26,
40 –47. (doi:10.1093/cvr/26.1.40)
Hamann M, Attwell D. 1996 Non-synaptic release
of ATP by electrical stimulation in slices of rat
hippocampus, cerebellum and habenula.
Eur. J. Neurosci. 8, 1510–1515. (doi:10.1111/j.
1460-9568.1996.tb01613.x)
Leybaert L, Braet K, Vandamme W, Cabooter L,
Martin PE, Evans WH. 2003 Connexin channels,
connexin mimetic peptides and ATP release. Cell.
Commun. Adhes. 10, 251–257. (doi:10.1080/cac.10.
4-6.251.257)
Locovei S, Bao L, Dahl G. 2006 Pannexin 1 in
erythrocytes: function without a gap. Proc. Natl
Acad. Sci. USA 103, 7655– 7659. (doi:10.1073/pnas.
0601037103)
Verkhratsky A, Burnstock G. 2014 Biology of
purinergic signalling: its ancient evolutionary roots,
its omnipresence and its multiple functional
significance. Bioessays 36, 697 –705. (doi:10.1002/
bies.201400024)
23. Ballerini P, Di Iorio P, Ciccarelli R, Nargi E, D’Alimonte I,
Traversa U, Rathbone MP, Caciagli F. 2002 Glial cells
express multiple ATP binding cassette proteins which
are involved in ATP release. Neuroreport 13, 1789–
1792. (doi:10.1097/00001756-200210070-00019)
24. Silverman W, Locovei S, Dahl G. 2008 Probenecid, a
gout remedy, inhibits pannexin 1 channels.
Am. J. Physiol. Cell Physiol. 295, C761–C767.
(doi:10.1152/ajpcell.00227.2008)
25. Boassa D, Ambrosi C, Qiu F, Dahl G, Gaietta G,
Sosinsky G. 2007 Pannexin1 channels contain a
glycosylation site that targets the hexamer to the
plasma membrane. J. Biol. Chem. 282, 31 733 –
31 743. (doi:10.1074/jbc.M702422200)
26. Dahl G, Locovei S. 2006 Pannexin: to gap or not to
gap, is that a question? IUBMB Life 58, 409 –419.
(doi:10.1080/15216540600794526)
27. Reisin IL et al. 1994 The cystic fibrosis
transmembrane conductance regulator is a dual ATP
and chloride channel. J. Biol. Chem. 269, 20 584–
20 591.
28. Liu HT, Toychiev AH, Takahashi N, Sabirov RZ, Okada
Y. 2008 Maxi-anion channel as a candidate pathway
for osmosensitive ATP release from mouse astrocytes
in primary culture. Cell Res. 18, 558 –565. (doi:10.
1038/cr.2008.49)
29. Cotrina ML, Lin JH, Lopez-Garcia JC, Naus CC,
Nedergaard M. 2000 ATP-mediated glia signaling.
J. Neurosci. 20, 2835–2844.
30. Taruno A et al. 2013 CALHM1 ion channel mediates
purinergic neurotransmission of sweet, bitter and
umami tastes. Nature 495, 223 –226. (doi:10.1038/
nature11906)
31. Bao L, Locovei S, Dahl G. 2004 Pannexin membrane
channels are mechanosensitive conduits for ATP.
FEBS Lett. 572, 65 –68. (doi:10.1016/j.febslet.2004.
07.009)
32. Grygorczyk R, Tabcharani JA, Hanrahan JW. 1996
CFTR channels expressed in CHO cells do not have
detectable ATP conductance. J. Membr. Biol. 151,
139–148. (doi:10.1007/s002329900065)
33. Wiszniewski L, Sanz J, Scerri I, Gasparotto E, Dudez
T, Lacroix JS, Suter S, Gallati S, Chanson M. 2007
Functional expression of connexin30 and connexin31
in the polarized human airway epithelium.
Differentiation 75, 382– 392. (doi:10.1111/j.14320436.2007.00157.x)
Phil. Trans. R. Soc. B 370: 20140191
References
9
rstb.royalsocietypublishing.org
in the same size range. Alternative roles of Panx1 may even
include non-channel functions of the protein.
The function of the other two pannexins (Panx2 and
Panx3) has remained elusive. Panx2 expression is almost
exclusively found in the nervous system. While the Panx2
protein is capable of forming a channel [64], no physiological
stimuli have been identified so far. Furthermore, its predominant localization in cytoplasmic compartments [107] suggests
an alternative function to that of Panx1.
Panx3 is similarly mysterious, because its localization in some
studies is also mainly intracellular [108,109]. Furthermore, the
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
63. Silverman WR, de Rivero Vaccari JP, Locovei S, Qiu F,
Carlsson SK, Scemes E, Keane RW, Dahl G. 2009 The
pannexin 1 channel activates the inflammasome
in neurons and astrocytes. J. Biol. Chem. 284,
18 143 –18 151. (doi:10.1074/jbc.M109.004804)
64. Ambrosi C, Gassmann O, Pranskevich JN, Boassa D,
Smock A, Wang J, Dahl G, Steinem C, Sosinsky GE.
2010 Pannexin1 and Pannexin2 channels show
quaternary similarities to connexons and different
oligomerization numbers from each other. J. Biol.
Chem. 285, 24 420–24 431. (doi:10.1074/jbc.M110.
115444)
65. Huang Y, Grinspan JB, Abrams CK, Scherer SS. 2007
Pannexin1 is expressed by neurons and glia but
does not form functional gap junctions. Glia 55,
46– 56. (doi:10.1002/glia.20435)
66. Cone AC, Ambrosi C, Scemes E, Martone ME,
Sosinsky GE. 2013 A comparative antibody analysis
of pannexin1 expression in four rat brain regions
reveals varying subcellular localizations. Front.
Pharmacol. 4, 6. (doi:10.3389/fphar.2013.00006)
67. Bunse S, Locovei S, Schmidt M, Qiu F, Zoidl G, Dahl
G, Dermietzel R. 2009 The potassium channel
subunit Kvbeta3 interacts with pannexin 1 and
attenuates its sensitivity to changes in redox
potentials. FEBS J. 276, 6258 –6270. (doi:10.1111/j.
1742-4658.2009.07334.x)
68. Thompson RJ, Jackson MF, Olah ME, Rungta RL,
Hines DJ, Beazely MA, MacDonald JF, MacVicar BA.
2008 Activation of pannexin-1 hemichannels
augments aberrant bursting in the hippocampus.
Science 322, 1555– 1559. (doi:10.1126/science.
1165209)
69. Billaud M, Sandilos JK, Isakson BE. 2012 Pannexin 1
in the regulation of vascular tone. Trends
Cardiovasc. Med. 22, 68– 72. (doi:10.1016/j.tcm.
2012.06.014)
70. Murali S, Zhang M, Nurse CA. 2014 Angiotensin II
mobilizes intracellular calcium and activates
pannexin-1 channels in rat carotid body type II cells
via AT1 receptors. J. Physiol. 592, 4747– 4762.
(doi:10.1113/jphysiol.2014.279299)
71. Shahidullah M, Mandal A, Beimgraben C, Delamere
NA. 2011 Hyposmotic stress causes ATP release and
stimulates Na,K-ATPase activity in porcine lens.
J. Cell Physiol. 227, 1428 –1437. (doi:10.1002/jcp.
22858)
72. Xia J, Lim JC, Lu W, Beckel JM, Macarak EJ, Laties
AM, Mitchell CH. 2012 Neurons respond directly to
mechanical deformation with pannexin-mediated
ATP release and autostimulation of P2X7 receptors.
J. Physiol. 590, 2285–2304. (doi:10.1113/jphysiol.
2012.227983)
73. Beckel JM et al. 2014 Mechanosensitive release of
adenosine 50 -triphosphate through pannexin channels
and mechanosensitive upregulation of pannexin
channels in optic nerve head astrocytes: a mechanism
for purinergic involvement in chronic strain. Glia 62,
1486–1501. (doi:10.1002/glia.22695)
74. Li A, Leung CT, Peterson-Yantorno K, Mitchell CH,
Civan MM. 2010 Pathways for ATP release by bovine
ciliary epithelial cells, the initial step in purinergic
regulation of aqueous humor inflow. Am. J. Physiol.
10
Phil. Trans. R. Soc. B 370: 20140191
48. Seminario-Vidal L et al. 2011 Rho signaling
regulates pannexin 1-mediated ATP release from
airway epithelia. J. Biol. Chem. 286, 26 277–
26 286. (doi:10.1074/jbc.M111.260562)
49. Qu Y, Misaghi S, Newton K, Gilmour LL, Louie S,
Cupp JE, Dubyak GR, Hackos D, Dixit VM. 2011
Pannexin-1 is required for ATP release during
apoptosis but not for inflammasome activation.
J. Immunol. 186, 6553–6561. (doi:10.4049/
jimmunol.1100478)
50. Li S, Bjelobaba I, Yan Z, Kucka M, Tomic M, Stojilkovic
SS. 2011 Expression and roles of pannexins in ATP
release in the pituitary gland. Endocrinology 152,
2342–2352. (doi:10.1210/en.2010-1216)
51. Pelegrin P, Surprenant A. 2006 Pannexin-1 mediates
large pore formation and interleukin-1beta release
by the ATP-gated P2X7 receptor. EMBO J. 25,
5071 –5082. (doi:10.1038/sj.emboj.7601378)
52. Locovei S, Wang J, Dahl G. 2006 Activation of
pannexin 1 channels by ATP through P2Y receptors
and by cytoplasmic calcium. FEBS Lett. 580,
239 –244. (doi:10.1016/j.febslet.2005.12.004)
53. Locovei S, Scemes E, Qiu F, Spray DC, Dahl G. 2007
Pannexin1 is part of the pore forming unit of the
P2X receptor death complex. FEBS Lett. 581,
483 –488. (doi:10.1016/j.febslet.2006.12.056)
54. Qiu F, Dahl G. 2009 A permeant regulating its
permeation pore: inhibition of pannexin 1 channels
by ATP. Am. J. Physiol. Cell Physiol. 296,
C250– C255. (doi:10.1152/ajpcell.00433.2008)
55. Qiu F, Wang J, Dahl G. 2012 Alanine substitution
scanning of pannexin1 reveals amino acid residues
mediating ATP sensitivity. Purinergic Signal. 8,
81 –90. (doi:10.1007/s11302-011-9263-6)
56. Wang J, Dahl G. 2010 SCAM analysis of Panx1
suggests a peculiar pore structure. J. Gen. Physiol.
136, 515 –527. (doi:10.1085/jgp.201010440)
57. Thompson RJ, Zhou N, MacVicar BA. 2006 Ischemia
opens neuronal gap junction hemichannels. Science
312, 924 –927. (doi:10.1126/science.1126241)
58. Wang J, Ambrosi C, Qiu F, Jackson DG, Sosinsky G,
Dahl G. 2014 The membrane protein Pannexin1
forms two open-channel conformations depending
on the mode of activation. Sci. Signal. 7, ra69.
(doi:10.1126/scisignal.2005431)
59. Jiang H, Zhu AG, Mamczur M, Falck JR, Lerea KM,
McGiff JC. 2007 Stimulation of rat erythrocyte P2X7
receptor induces the release of epoxyeicosatrienoic
acids. Br. J. Pharmacol. 151, 1033 –1040. (doi:10.
1038/sj.bjp.0707311)
60. Santiago MF VJ, Patel NK, Lutz SE, Caille D,
Charollais A, Meda P, Scemes E. 2011 Targeting
Pannexin1 improves seizure outcome. PLoS ONE 6,
e25178. (doi:10.1371/journal.pone.0025178)
61. Karatas H, Erdener SE, Gursoy-Ozdemir Y, Lule S,
Eren-Kocak E, Sen ZD, Dalkara T. 2013 Spreading
depression triggers headache by activating neuronal
Panx1 channels. Science 339, 1092 –1095. (doi:10.
1126/science.1231897)
62. Gulbransen BD et al. 2012 Activation of neuronal
P2X7 receptor-pannexin-1 mediates death of enteric
neurons during colitis. Nat. Med. 18, 600 –604.
(doi:10.1038/nm.2679)
rstb.royalsocietypublishing.org
34. Bruzzone R, Barbe MT, Jakob NJ, Monyer H. 2005
Pharmacological properties of homomeric and
heteromeric pannexin hemichannels expressed in
Xenopus oocytes. J. Neurochem. 92, 1033 –1043.
(doi:10.1111/j.1471-4159.2004.02947.x)
35. Dahl G, Qiu F, Wang J. 2013 The bizarre
pharmacology of the ATP release channel
pannexin1. Neuropharmacology 75, 150 –159.
(doi:10.1016/j.neuropharm.2013.02.019)
36. Panchin Y, Kelmanson I, Matz M, Lukyanov K, Usman
N, Lukyanov S. 2000 A ubiquitous family of putative
gap junction molecules. Curr. Biol. 10, R473–R474.
(doi:10.1016/S0960-9822(00) 00576-5)
37. Ransford GA, Fregien N, Qiu F, Dahl G, Conner GE,
Salathe M. 2009 Pannexin 1 contributes to ATP
release in airway epithelia. Am. J. Respir. Cell Mol.
Biol. 41, 525–534. (doi:10.1165/rcmb.20080367OC)
38. Hanner F, Lam L, Nguyen MT, Yu A, Peti-Peterdi J.
2012 Intrarenal localization of the plasma
membrane ATP channel pannexin1. Am. J. Physiol.
Renal Physiol. 303, F1454–F1459. (doi:10.1152/
ajprenal.00206.2011)
39. Penuela S, Bhalla R, Gong XQ, Cowan KN, Celetti SJ,
Cowan BJ, Bai D, Shao Q, Laird DW. 2007 Pannexin
1 and pannexin 3 are glycoproteins that exhibit
many distinct characteristics from the connexin
family of gap junction proteins. J. Cell Sci. 120,
3772–3783. (doi:10.1242/jcs.009514)
40. Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H.
2003 Pannexins, a family of gap junction proteins
expressed in brain. Proc. Natl Acad. Sci. USA 100,
13 644–13 649. (doi:10.1073/pnas.2233464100)
41. Wang J, Ma M, Locovei S, Keane RW, Dahl G. 2007
Modulation of membrane channel currents by gap
junction protein mimetic peptides: size matters.
Am. J. Physiol. Cell Physiol. 293, C1112 –C1119.
(doi:10.1152/ajpcell.00097.2007)
42. Bao L, Samuels S, Locovei S, Macagno ER, Muller KJ,
Dahl G. 2007 Innexins form two types of channels.
FEBS Lett. 581, 5703– 5708. (doi:10.1016/j.febslet.
2007.11.030)
43. Samuels SE, Lipitz JB, Dahl G, Muller KJ. 2010
Neuroglial ATP release through innexin channels
controls microglial cell movement to a nerve injury.
J. Gen. Physiol. 136, 425–442. (doi:10.1085/jgp.
201010476)
44. Samuels SE, Lipitz JB, Wang J, Dahl G, Muller KJ.
2013 Arachidonic acid closes innexin/pannexin
channels and thereby inhibits microglia cell
movement to a nerve injury. Dev. Neurobiol. 73,
621–631. (doi:10.1002/dneu.22088)
45. Dahl G, Muller KJ. 2014 Innexin and pannexin
channels and their signaling. FEBS Lett. 588,
1396–1402. (doi:10.1016/j.febslet.2014.03.007)
46. Qiu F, Wang J, Spray DC, Scemes E, Dahl G. 2011
Two non-vesicular ATP release pathways in the
mouse erythrocyte membrane. FEBS Lett. 585,
3430–3435. (doi:10.1016/j.febslet.2011.09.033)
47. Suadicani SO, Iglesias R, Wang J, Dahl G, Spray DC,
Scemes E. 2012 ATP signaling is deficient in
cultured pannexin1-null mouse astrocytes. Glia 60,
1106–1116. (doi:10.1002/glia.22338)
Downloaded from http://rstb.royalsocietypublishing.org/ on June 18, 2017
76.
78.
79.
80.
81.
82.
83.
84.
85.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
Channels (Austin) 8, 103–109. (doi:10.4161/chan.
27545)
Poon IK, Chiu YH, Armstrong AJ, Kinchen JM,
Juncadella IJ, Bayliss DA, Ravichandran KS. 2014
Unexpected link between an antibiotic, pannexin
channels and apoptosis. Nature 507, 329 –334.
(doi:10.1038/nature13147)
Sandilos JK, Chiu YH, Chekeni FB, Armstrong AJ,
Walk SF, Ravichandran KS, Bayliss DA. 2012
Pannexin 1, an ATP release channel, is activated by
caspase cleavage of its pore-associated C-terminal
autoinhibitory region. J. Biol. Chem. 287, 11 303–
11 311. (doi:10.1074/jbc.M111.323378)
MacVicar BA, Thompson RJ. 2010 Non-junction
functions of pannexin-1 channels. Trends Neurosci.
33, 93 – 102. (doi:10.1016/j.tins.2009.11.007)
Ma W, Compan V, Zheng W, Martin E, North RA,
Verkhratsky A, Surprenant A. 2012 Pannexin 1 forms
an anion-selective channel. Pflugers Arch. 463,
585–592. (doi:10.1007/s00424-012-1077-z)
Romanov RA, Bystrova MF, Rogachevskaya OA,
Sadovnikov VB, Shestopalov VI, Kolesnikov SS. 2012
The ATP permeability of pannexin 1 channels in a
heterologous system and in mammalian taste cells
is dispensable. J. Cell Sci. 125, 5514– 5523. (doi:10.
1242/jcs.111062)
Nualart-Marti A et al. 2013 Role of connexin 32
hemichannels in the release of ATP from peripheral
nerves. Glia 61, 1976 –1989. (doi:10.1002/glia.
22568)
Hansen DB, Ye ZC, Calloe K, Braunstein TH,
Hofgaard JP, Ransom BR, Nielsen MS, MacAulay N.
2014 Activation, permeability, and inhibition of
astrocytic and neuronal large pore (hemi)channels.
J. Biol. Chem. 289, 26 058– 26 073. (doi:10.1074/
jbc.M114.582155)
Wang J, Jackson DG, Dahl G. 2013 The food dye
FD&C Blue No. 1 is a selective inhibitor of the ATP
release channel Panx1. J. Gen. Physiol. 141,
649–656. (doi:10.1085/jgp.201310966)
Le Vasseur M, Lelowski J, Bechberger JF, Sin WC,
Naus CC. 2014 Pannexin 2 protein expression is not
restricted to the CNS. Front. Cell Neurosci. 8, 392.
(doi:10.3389/fncel.2014.00392)
Iwamoto T, Nakamura T, Doyle A, Ishikawa M, de
Vega S, Fukumoto S, Yamada Y. 2010 Pannexin 3
regulates intracellular ATP/cAMP levels and
promotes chondrocyte differentiation. J. Biol. Chem.
285, 18 948 –18 958. (doi:10.1074/jbc.M110.
127027)
Ishikawa M, Iwamoto T, Nakamura T, Doyle A,
Fukumoto S, Yamada Y. 2011 Pannexin 3 functions
as an ER Ca2þ channel, hemichannel, and gap
junction to promote osteoblast differentiation. J. Cell
Biol. 193, 1257 –1274. (doi:10.1083/jcb.201101050)
Baranova A et al. 2004 The mammalian pannexin
family is homologous to the invertebrate innexin
gap junction proteins. Genomics 83, 706–716.
(doi:10.1016/j.ygeno.2003.09.025)
11
Phil. Trans. R. Soc. B 370: 20140191
77.
86. Dahl G, Keane RW. 2012 Pannexin: from discovery
to bedside in 11+4 years? Brain Res. 1487,
150 –159. (doi:10.1016/j.brainres.2012.04.058)
87. Weilinger NL, Tang PL, Thompson RJ. 2012 Anoxiainduced NMDA receptor activation opens pannexin
channels via Src family kinases. J. Neurosci. 32,
12 579– 12 588. (doi:10.1523/JNEUROSCI.126712.2012)
88. Isakson BE, Thompson RJ. 2014 Pannexin-1 as a
potentiator of ligand-gated receptor signaling. Channels
(Austin) 8, 118–123. (doi:10.4161/chan.27978)
89. Seminario-Vidal L, Kreda S, Jones L, O’Neal W, Trejo
J, Boucher RC, Lazarowski ER. 2009 Thrombin
promotes release of ATP from lung epithelial cells
through coordinated activation of Rho- and Ca2þdependent signaling pathways. J. Biol. Chem. 284,
20 638– 20 648. (doi:10.1074/jbc.M109.004762)
90. Pinheiro AR, Paramos-de-Carvalho D, Certal M, Costa
C, Magalhaes-Cardoso MT, Ferreirinha F, Costa M,
Correia-de-Sá P. 2013 Bradykinin-induced Ca2þ
signaling in human subcutaneous fibroblasts
involves ATP release via hemichannels leading to
P2Y12 receptors activation. Cell Commun. Signal. 11,
70. (doi:10.1186/1478-811X-11-70)
91. Pinheiro AR et al. 2013 Histamine induces ATP
release from human subcutaneous fibroblasts,
via pannexin-1 hemichannels, leading to Ca2þ
mobilization and cell proliferation. J. Biol. Chem.
288, 27 571–27 583. (doi:10.1074/jbc.M113.
460865)
92. da Silva-Souza HA, de Lira MN, Patel NK, Spray DC,
Persechini PM, Scemes E. 2014 Inhibitors of the
5-lipoxygenase pathway activate pannexin1
channels in macrophages via the thromboxane
receptor. Am. J. Physiol. Cell Physiol. 307,
C571– C579. (doi:10.1152/ajpcell.00087.2014)
93. Momboisse F et al. 2014 Pannexin 1 channels: new
actors in the regulation of catecholamine release
from adrenal chromaffin cells. Front. Cell Neurosci. 8,
270. (doi:10.3389/fncel.2014.00270)
94. Jackson DG, Wang J, Keane RW, Scemes E, Dahl G.
2014 ATP and potassium ions: a deadly combination
for astrocytes. Sci. Rep. 4, 4576. (doi:10.1038/
srep04576)
95. Chekeni FB et al. 2010 Pannexin 1 channels mediate
‘find-me’ signal release and membrane permeability
during apoptosis. Nature 467, 863– 867. (doi:10.
1038/nature09413)
96. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y,
Jung S, Littman DR, Dustin ML, Gan W-B. 2005 ATP
mediates rapid microglial response to local brain
injury in vivo. Nat. Neurosci. 8, 752– 758. (doi:10.
1038/nn1472)
97. Duan Y, Sahley CL, Muller KJ. 2009 ATP and NO
dually control migration of microglia to nerve
lesions. Dev. Neurobiol. 69, 60 –72. (doi:10.1002/
dneu.20689)
98. Chiu YH, Ravichandran KS, Bayliss DA. 2014 Intrinsic
properties and regulation of Pannexin 1 channel.
rstb.royalsocietypublishing.org
75.
Cell Physiol. 299, C1308– C1317. (doi:10.1152/
ajpcell.00333.2010)
Islam MR, Uramoto H, Okada T, Sabirov RZ, Okada Y.
2012 Maxi-anion channel and pannexin 1
hemichannel constitute separate pathways for
swelling-induced ATP release in murine L929
fibrosarcoma cells. Am. J. Physiol. Cell Physiol. 303,
C924–C935. (doi:10.1152/ajpcell.00459.2011)
Sridharan M, Adderley SP, Bowles EA, Egan TM,
Stephenson AH, Ellsworth ML, Sprague RS.
2010 Pannexin 1 is the conduit for low oxygen
tension-induced ATP release from human
erythrocytes. Am. J. Physiol. Heart Circ. Physiol.
299, H1146– H1152. (doi:10.1152/ajpheart.
00301.2010)
Tang XD, Xu R, Reynolds MF, Garcia ML, Heinemann
SH, Hoshi T. 2003 Haem can bind to and inhibit
mammalian calcium-dependent Slo1 BK channels.
Nature 425, 531 –535. (doi:10.1038/nature02003)
Zhang M, Piskuric NA, Vollmer C, Nurse CA. 2012
P2Y2 receptor activation opens pannexin-1 channels
in rat carotid body type II cells: potential role in
amplifying the neurotransmitter ATP. J. Physiol.
590, 4335 –4350. (doi:10.1113/jphysiol.2012.
236265)
Ma W, Hui H, Pelegrin P, Surprenant A. 2009
Pharmacological characterization of pannexin-1
currents expressed in mammalian cells.
J. Pharmacol. Exp. Ther. 328, 409–418. (doi:10.
1124/jpet.108.146365)
Hung SC, Choi CH, Said-Sadier N, Johnson L,
Atanasova KR, Sellami H, Yilmaz Ö, Ojcius DM. 2013
P2X4 assembles with P2X7 and pannexin-1 in
gingival epithelial cells and modulates ATP-induced
reactive oxygen species production and
inflammasome activation. PLoS ONE 8, e70210.
(doi:10.1371/journal.pone.0070210)
Li S, Tomic M, Stojilkovic SS. 2011 Characterization
of novel Pannexin 1 isoforms from rat pituitary cells
and their association with ATP-gated P2X channels.
Gen. Comp. Endocrinol. 174, 202–210. (doi:10.
1016/j.ygcen.2011.08.019)
Iglesias R, Locovei S, Roque A, Alberto AP, Dahl G,
Spray DC, Scemes E. 2008 P2X7 receptor–Pannexin1
complex: pharmacology and signaling. Am. J. Physiol.
Cell Physiol. 295, C752–C760. (doi:10.1152/ajpcell.
00228.2008)
Di Virgilio F et al. 2001 Nucleotide receptors: an
emerging family of regulatory molecules in blood
cells. Blood 97, 587– 600. (doi:10.1182/blood.V97.
3.587)
Ballerini P et al. 1996 Rat astroglial P2Z (P2X7)
receptors regulate intracellular calcium and purine
release. Neuroreport 7, 2533 –2537. (doi:10.1097/
00001756-199611040-00026)
Bodin P, Burnstock G. 1996 ATP-stimulated release
of ATP by human endothelial cells. J. Cardiovasc.
Pharmacol. 27, 872– 875. (doi:10.1097/00005344199606000-00015)